The present invention relates to a novel family of purified proteins designated as Bone Morphogenetic Proteins (BMP)-17 and BMP-18, related proteins, DNA encoding them, and processes for obtaining them. These proteins may be used to induce bone and/or cartilage or other connective tissue formation, and in wound healing and tissue repair. These proteins may also be used for augmenting the activity of other bone morphogenetic proteins.
The search for the molecule or molecules responsible for the bone-, cartilage-, and other connective tissue-inductive activity present in bone and other tissue extracts has led to the discovery of a novel set of molecules called the Bone Morphogenetic Proteins (BMPs). The structures of several proteins, designated BMP-1 through BMP-16 have previously been elucidated. The unique inductive activities of these proteins, along with their presence in bone, suggests that they are important regulators of bone repair processes, and may be involved in the normal maintenance of bone tissue. There is a need to identify whether additional proteins, particularly human proteins, exist which play a role in these processes. The present invention relates to the identification of such a novel human protein, which the inventors have designated human BMP-17 and BMP-18.
Human BMP-17 and BMP-18 appear to be human homologs of a murine protein called Lefty. The nucleotide and amino acid sequences of murine Lefty are described in Zhou et al., Nature, 361:543-547 (1993). The murine Lefty gene has been described as being expressed in the mouse node during gastrulation. A related human protein, designated endometrial bleeding associated factor [EBAF] was published in Kothapelli et al., J. Clin. Invest., 99:2342-2350 (1997).
As used herein, the term BMP-17 and BMP-18 proteins refer to the human BMP-17 and BMP-18 proteins, having the amino acid sequences specified in SEQUENCE (SEQ) ID NO:2 and SEQ ID NO: 4, as well as DNA sequences encoding the BMP-17 and BMP-18 proteins, such as the native human sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3. Also included are naturally occurring allelic sequences of SEQ ID NO:1 and 3, and equivalent degenerative codon sequences of the above.
The BMP-17 (SEQ ID NO: 1) and BMP-18 (SEQ ID NO: 3) DNA sequences and amino acid sequences (SEQ ID NO: 2 and 4, respectively) are set forth in the Sequence Listings. BMP-17 and BMP-18 proteins may be capable of inducing the formation of cartilage, bone, or other connective tissue, or combinations thereof. The cartilage and/or bone and/or other connective tissue formation activity in the rat bone formation assay described below. BMP-17 and BMP-18 proteins may be further characterized by the ability to demonstrate effects upon the growth and/or differentiation of embryonic cells and/or stem cells. Thus, the proteins or compositions of the present invention may also be useful for treating cell populations, such as embryonic cells or stem cell populations, to enhance or enrich the growth and/or differentiation of the cells. Alternatively, the proteins or compositions of the present invention may also be useful for maintenance of a cell population, including differentiated cell populations, for example, neuronal cells, epithelial cells, dendritic cells, chondrocytes, osteocytes, muscle cells or cells of other differentiated phenotypes.
Human BMP-17 proteins may be produced by culturing a cell transformed with a DNA sequence comprising nucleotide a DNA sequence encoding the mature BMP-17 polypeptide, comprising nucleotide #427 to nucleotide #1098 as shown in SEQ ID NO: 1, and recovering and purifying from the culture medium a protein characterized by the amino acid sequence comprising amino acids #1 to #224 as shown in SEQ ID NO:2 substantially free from other proteinaceous materials with which it is co-produced. For production in mammalian cells, the DNA sequence further comprises a DNA sequence encoding a suitable propeptide 5xe2x80x2 to and linked in frame to the nucleotide sequence encoding the mature BMP-17-related polypeptide.
Human BMP-18 proteins may be produced by culturing a cell transformed with a DNA sequence comprising nucleotide a DNA sequence encoding the mature BMP-18 polypeptide, comprising nucleotide #406 to nucleotide #1098 as shown in SEQ ID NO: 3, and recovering and purifying from the culture medium a protein characterized by the amino acid sequence comprising amino acids #1 to #231 as shown in SEQ ID NO:4 substantially free from other proteinaceous materials with which it is co-produced. For production in mammalian cells, the DNA sequence further comprises a DNA sequence encoding a suitable propeptide 5xe2x80x2 to and linked in frame to the nucleotide sequence encoding the mature BMP-18-related polypeptide.
The propeptide may be the native BMP-17 or BMP-18-related propeptide, or may be a propeptide from another protein of the TGF-xcex2 superfamily. Where the native BMP-17 and BMP-18 propeptide is used, human BMP-17 and BMP-18 may be produced by culturing a cell transformed with a DNA sequence comprising a DNA sequence encoding the full BMP-17 and BMP-18 polypeptide, comprising nucleotides #1 to #1098 as shown in SEQ ID NO: 1, or nucleotides #1 to #1098 as shown in SEQ ID NO: 3, producing a protein characterized by the amino acid sequence comprising amino acids #xe2x88x92142 to #224 as shown in SEQ ID NO:2, of which amino acids #xe2x88x92142 to xe2x88x921 comprise the native propeptide of human BMP-17; or #xe2x88x92135 to #231 of SEQ ID NO: 4 of which amino acids #xe2x88x92135 to xe2x88x921 comprise the native propeptide of human BMP-18, and recovering and purifying from the culture medium a protein characterized by the amino acid sequence comprising amino acids #1 to #224 as shown in SEQ ID NO:2, or #1 to #231 of SEQ ID NO:4, respectively, substantially free from other proteinaceous materials with which it is co-produced.
Based in part upon the Von Heginje signal peptide prediction algorithm, approximately the first 17 to 23 amino acids of SEQ ID NO: 2 and 4 appear to be involved in signalling for the secretion of the mature peptide. Accordingly, in one embodiment of the invention, DNA encoding a signal peptide,such as the native BMP-17 or BMP-18 signal peptide, or another recognized signal peptide, may be linked directly to the sequence encoding the mature BMP-17 or BMP-18 peptide.
It is expected that other species, particularly human, have DNA sequences homologous to human BMP-17 and BMP-18 protein. The invention, therefore, includes methods for obtaining the DNA sequences encoding human BMP-17 and BMP-18 proteins, the DNA sequences obtained by those methods, and the human proteins encoded by those DNA sequences. This method entails utilizing the human BMP-17 and BMP-18 nucleotide sequences or portions thereof to design probes to screen libraries for the corresponding gene from other species or coding sequences or fragments thereof from using standard techniques. Thus, the present invention may include DNA sequences from other species, which are homologous to human BMP-17 and BMP-18 proteins and can be obtained using the human BMP-17 and/or BMP-18 sequences. The present invention may also include functional fragments of the human BMP-17 and BMP-18 proteins, and DNA sequences encoding such functional fragments, as well as functional fragments of other related proteins. The ability of such a fragment to function is determinable by assay of the protein in the biological assays described for the assay of the BMP-17 and BMP-18 proteins. DNA sequences encoding the complete mature human BMP-17 (SEQ ID NO: 1 and BMP-18 protein (SEQ ID NO:3) and the corresponding amino acid sequences (SEQ ID NO:2 and 4, respectively) are set forth herein. The BMP-17 and BMP-18 proteins of the present invention, such as human BMP-17 and BMP-18, may be produced by culturing a cell transformed with the correlating DNA sequence, such as the human BMP-17 and BMP-18 DNA sequence, and recovering and purifying protein, such as BMP-17 or BMP-18, from the culture medium. The purified expressed protein is substantially free from other proteinaceous materials with which it is co-produced, as well as from other contaminants. The recovered purified protein is contemplated to exhibit cartilage and/or bone and/or connective tissue formation activity. Thus, the proteins of the invention may be further characterized by the ability to demonstrate cartilage and/or bone and/or other connective tissue formation activity in the rat bone formation assay described below. BMP-17 and BMP-18 proteins may be further characterized by the ability to demonstrate effects upon the growth and/or differentiation of embryonic cells and/or stem cells. Thus, the proteins or compositions of the present invention may also be characterized by their ability to enhance or enrich the growth and/or differentiation of the cells.
Another aspect of the invention provides pharmaceutical compositions containing a therapeutically effective amount of human BMP-17 and/or BMP-18 protein, in a pharmaceutically acceptable vehicle or carrier. These compositions of the invention may be used in the formation of bone. These compositions may further be utilized for the formation of cartilage, or other connective tissue, including tendon, ligament, meniscus and other connective tissue, as well as combinations of the above, for example regeneration of the tendon-to-bone attachment apparatus. The compositions of the present invention, such as compositions of human BMP-17 and/or BMP-18, may also be used for wound healing and tissue repair. Compositions of the invention may further include at least one other therapeutically useful agent such as the BMP proteins BMP-1, BMP-2, BMP-3, BMP4, BMP-5, BMP-6 and BMP-7, disclosed for instance in U.S. Pat. Nos.5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publication WO91/18098; and BMP-9, disclosed in PCT publication WO93/00432, BMP-10, disclosed in U.S. Pat. No. 5,637,480; BMP-11, disclosed in U.S. Pat. No. 5,639,638, or BMP-12 or BMP-13, disclosed in U.S. Pat. No. 5,658,882, BMP-15, disclosed U.S. Pat. No. 5,635,372 and BMP-16, disclosed in co-pending patent application Ser. No. 08/715,202. Other compositions which may also be useful include Vgr-2, and any of the growth and differentiation factors [GDFs], including those described in PCT applications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others. Also useful in the present invention may be BIP, disclosed in WO94/01557; HP00269, disclosed in JP Publication number: 7-250688; and MP52, disclosed in PCT application WO93/16099. The disclosures of all of the above applications are hereby incorporated by reference.
The compositions of the invention may comprise, in addition to a BMP-17 and/or -18-related protein, other therapeutically useful agents including growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF-xcex1 and TGF-xcex2), activins, inhibins, and insulin-like growth factor (IGF). The compositions may also include an appropriate matrix for instance, for supporting the composition and providing a surface for bone and/or cartilage and/or other connective tissue growth. The matrix may provide slow release of the osteoinductive protein and/or the appropriate environment for presentation thereof.
The BMP-17 and/or BMP-18 containing compositions may be employed in methods for treating a number of bone and/or cartilage and/or other connective tissue defects, periodontal disease and healing of various types of tissues and wounds. The tissue and wounds which may be treated include epidermis, nerve, muscle, including cardiac muscle, and other tissues and wounds, and other organs such as liver, lung, epithelium, brain, spleen, cardiac, pancreas and kidney tissue. These methods, according to the invention, entail administering to a patient needing such bone and/or cartilage and/or other connective tissue formation, wound healing or tissue repair, an effective amount of a BMP-17 and/or BMP-18 protein. The BMP-17 and/or BMP-18 containing compositions may also be used to treat or prevent such conditions as osteoarthritis, osteoporosis, and other abnormalities of bone, cartilage, muscle, tendon, ligament or other connective tissue, organs such as liver, lung, epithelium, brain, spleen, cardiac, pancreas and kidney tissue, and other tissues. These methods may also entail the administration of a protein of the invention in conjunction with at least one other BMP protein as described above. In addition, these methods may also include the administration of a BMP-17 and/or BMP-18 protein with other growth factors including EGF, FGF, TGF-xcex1, TGF-xcex2, activin, inhibin and IGF.
Still a further aspect of the invention are DNA sequences coding for expression of a BMP-17 and/or BMP-18 protein. Such sequences include the sequence of nucleotides in a 5xe2x80x2 to 3xe2x80x2 direction illustrated in SEQ ID NO: 1 or SEQ ID NO: 3, DNA sequences which, but for the degeneracy of the genetic code, are identical to the DNA sequence SEQ ID NO: 1 or SEQ ID NO: 3, and encode the protein of SEQ ID NO: 2 or SEQ ID NO: 4. Further included in the present invention are DNA sequences which hybridize under stringent conditions with the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and encode a protein having the ability to induce formation of cartilage, bone and/or other connective tissue, organs such as liver, lung, epithelium, brain, spleen, cardiac, pancreas and kidney tissue, or other activities disclosed for BMP-17 and BMP-18. Preferred DNA sequences include those which hybridize under stringent conditions [For example, see conditions described in Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to 389]. It is generally preferred that such DNA sequences encode a polypeptide which is at least about 80% homologous, and more preferably at least about 90% homologous, to the conserved C-terminal cysteine structure of the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO: 4. Finally, allelic or other variations of the sequences of SEQ ID NO: 1 or SEQ ID NO: 3, whether or not such nucleotide changes result in changes in the peptide sequence, where the peptide sequence retains one or more BMP-17 and/or BMP-18 activity, are also included in the present invention. The present invention also includes fragments of the DNA sequence of BMP-17 and/or BMP-18 shown in SEQ ID NO: 1 or SEQ ID NO: 3 which encode a polypeptide which retains the activity of BMP-17 and/or BMP-18 protein.
The DNA sequences of the present invention are useful, for example, as probes for the detection of mRNA encoding BMP-17 and/or BMP-18 in a given cell population. Thus, the present invention includes methods of detecting or diagnosing genetic disorders involving the BMP-17 and/or BMP-18 gene, or disorders involving cellular, organ or tissue disorders in which BMP-17 and/or BMP-18 is irregularly transcribed or expressed. The DNA sequences may also be useful for preparing vectors for gene therapy applications as described below.
A further aspect of the invention includes vectors comprising a DNA sequence as described above in operative association with an expression control sequence therefor. These vectors may be employed in a novel process for producing a BMP-17 and/or BMP-18 protein of the invention in which a cell line transformed with a DNA sequence encoding a BMP-17 and/or BMP-18 protein in operative association with an expression control sequence therefor, is cultured in a suitable culture medium and a BMP-17 and/or BMP-18-related protein is recovered and purified therefrom. This process may employ a number of known cells both prokaryotic and eukaryotic as host cells for expression of the polypeptide. The vectors may be used in gene therapy applications. In such use, the vectors may be transfected into the cells of a patient ex vivo, and the cells may be reintroduced into a patient. Alternatively, the vectors may be introduced into a patient in vivo through targeted transfection.
Still a further aspect of the invention are BMP-17 and/or BMP-18 proteins or polypeptides. Such polypeptides are characterized by having an amino acid sequence including the sequence illustrated in SEQ ID NO: 2 or SEQ ID NO: 4, variants of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, including naturally occurring allelic variants, and other variants in which the protein retains the ability to induce the formation of cartilage and/or bone and/or other connective tissue, or other organs such as liver, lung, epithelium, brain, spleen, cardiac, pancreas and kidney tissue, or other activity characteristic of BMP-17 and/or BMP-18. Preferred polypeptides include a polypeptide which is at least about 80% homologous, and more preferably at least about 90% homologous, to the mature human BMP-17 and/or BMP-18 amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO: 4. Finally, allelic or other variations of the sequences of SEQ ID NO: 2 or SEQ ID NO: 4, whether such amino acid changes are induced by mutagenesis, chemical alteration, or by alteration of DNA sequence used to produce the polypeptide, where the peptide sequence still has BMP-17 and/or BMP-18 activity, are also included in the present invention. The present invention also includes fragments of the amino acid sequence of BMP-17 and/or BMP-18 shown in SEQ ID NO: 2 or SEQ ID NO: 4 which retain the activity of BMP-17 and/or BMP-18 protein.
The purified proteins of the present inventions may be used to generate antibodies, either monoclonal or polyclonal, to human BMP-17 and/or BMP-18 and/or other BMP-17 and/or BMP-18-related proteins, using methods that are known in the art of antibody production. Thus, the present invention also includes antibodies to human BMP-17 and/or BMP-18 and/or other related proteins. The antibodies may be useful for purification of BMP-17 and/or BMP-18 and/or other BMP-17 and/or BMP-18 related proteins, or for inhibiting or preventing the effects of BMP-17 and/or BMP-18 related proteins. The BMP-17 and/or BMP-18 protein and related proteins may be useful for inducing the growth and/or differentiation of embryonic cells and/or stem cells. Thus, the proteins or compositions of the present invention may also be useful for treating relatively undifferentiated cell populations, such as embryonic cells or stem cell populations, to enhance or enrich the growth and/or differentiation of the cells. The treated cell populations may be useful for implantation and for gene therapy applications.
SEQ ID NO:1 is a nucleotide sequence containing nucleotide sequence encoding the entire mature human BMP-17 polypeptide.
SEQ ID NO:2 is a amino acid sequence containing the mature human BMP-17 polypeptide sequence.
SEQ ID NO: 3 is a nucleotide sequence containing nucleotide sequence encoding the entire mature human BMP-18 polypeptide.
SEQ ID NO: 4 is a amino acid sequence containing the mature human BMP-17 polypeptide sequence.
The human BMP-17 and BMP-18 sequences of the present invention may be obtained using the whole or fragments of the murine Lefty DNA sequence, or a partial human BMP-17 or BMP-18 sequence, as a probe. Thus, the human BMP-17 and BMP-18 DNA sequence comprise the DNA sequence of nucleotides #1 to #1098 of SEQ ID NO: 1 or #1 to #1098 of SEQ ID NO:3. The human BMP-17 and BMP-18 proteins comprise the sequences of amino acids #xe2x88x92142 to #224 of SEQ ID NO: 2, or #xe2x88x92135 to #231 of SEQ ID NO: 4, respectively. The mature human BMP-17 and BMP-18 proteins are encoded by nucleotides #427 to #1098 of SEQ ID NO:1 and #406 to #1098 of SEQ ID NO:3, respectively, and comprises the sequence of amino acids #1 to #224 of SEQ ID NO:2, or #1 to #231 of SEQ ID NO: 4, respectively.
It is expected that human BMP-17 and BMP-18 polypeptides, as expressed by mammalian cells such as CHO cells, exists as a heterogeneous population of active species of BMP-17 and BMP-18 proteins with varying N-termini. It is expected that active species will comprise an amino acid sequence beginning with the residue at amino acid #109 or #124 of SEQ ID NO:2 or #116 or #131 of SEQ ID NO: 4, respectively, or will comprise additional amino acid sequence further in the N-terminal direction. Thus, it is expected that DNA sequences encoding active BMP-17 and BMP-18 polypeptides will comprise a nucleotide sequence comprising nucleotides #1, #232, #406, #427, #751 or 190 796 to #1059 or #1098 of SEQ ID NO: 1, or #1, #232, #406, #427, #751 or #796 to #1059 or #1098 of SEQ. ID NO: 3, respectively. Accordingly, active species of human BMP-17 and BMP-18 are expected to include those comprising amino acids #xe2x88x92142, #xe2x88x9265, #xe2x88x927, #1, #109 or #124 to #211 or #224 of SEQ ID NO:2, or #xe2x88x92135, #xe2x88x9258, #1, #8, #116 or #131 to #218 or #231 of Seq ID NO:4, respectively.
A host cell may be transformed with a coding sequence encoding a propeptide suitable for the secretion of proteins by the host cell is linked in proper reading frame to the coding sequence for the mature BMP-17 and BMP-18 protein. For example, see U.S. Pat. No. 5,658,882, in which the propeptide of BMP-2 is fused to the DNA encoding a mature BMP-12 protein. The disclosure of this reference is hereby incorporated by reference. Thus, the present invention includes chimeric DNA molecules comprising a DNA sequence encoding a propeptide from a member of the TGF-xcex2 superfamily of proteins, other than BMP-17 and BMP-18, is linked in correct reading frame to a DNA sequence encoding human BMP-17 or BMP-18 protein, or a related protein. The term xe2x80x9cchimericxe2x80x9d is used to signify that the propeptide originates from a different polypeptide than the native BMP-17 or BMP-18 protein.
The N-terminus of one active species of human BMP-17 is expected to be experimentally determined by expression in E. coli to be as follows: [M]ARVTV. Thus, it appears that the N-terminus of this species of BMP-17 is at amino acid #1 of SEQ ID NO: 1, and a DNA sequence encoding said species of BMP-17 would comprise nucleotides #427 to #1098 of SEQ ID NO: 1. The apparent molecular weight of human BMP-17 monomer is expected to be experimentally determined by SDS-PAGE to be approximately 24.8 kD on a Novex 16% tricine gel. The human BMP-17 protein is expected to exist as a clear, colorless solution in 0.1% trifluoroacetic acid.
It is expected that other BMP-17 proteins, as expressed by mammalian cells such as CHO cells, also exist as a heterogeneous population of active species of BMP-17-related protein with varying N-termini. For example, it is expected that active species of human BMP-17 protein will comprise an amino acid sequence beginning with the cysteine residue at amino acid #109 or the glutamic acid residue at position #124 of SEQ ID NO 2 or will comprise additional amino acid sequence further in the N-terminal direction. Thus, it is expected that DNA sequences encoding active BMP-17 proteins include those which comprise a nucleotide sequence comprising nucleotides #1, 232, 406, 427, 751 or 796 to #1059 or 1098 of SEQ ID NO: 1. Accordingly, active human BMP-17 proteins include those comprising amino acids #xe2x88x92142, xe2x88x9265, xe2x88x927, 1, 109 or 124 to #211 or 224 of SEQ ID NO: 2.
The N-terminus of one active species of human BMP-18 is expected to be experimentally determined by expression in E. coli to be as follows: [M]LSPRS. Thus, it appears that the N-terminus of this species of BMP-18 is at amino acid #1 of SEQ ID NO: 3, and a DNA sequence encoding said species of BMP-18 would comprise nucleotides #406 to #1098 of SEQ ID NO:3. The apparent molecular weight of human BMP-18 monomer is expected to be experimentally determined by SDS-PAGE to be approximately 25.6 kD on a Novex 16% tricine gel. The human BMP-18 protein is expected to exist as a clear, colorless solution in 0.1% trifluoroacetic acid.
It is expected that other BMP-18 proteins, as expressed by mammalian cells such as CHO cells, also exist as a heterogeneous population of active species of BMP-18-related protein with varying N-termini. For example, it is expected that active species of human BMP-18 protein will comprise an amino acid sequence beginning with the cysteine residue at amino acid #116 or the glutamic acid residue at position #131 of SEQ ID NO:4 or will comprise additional amino acid sequence further in the N-terminal direction. Thus, it is expected that DNA sequences encoding active BMP-18 proteins include those which comprise a nucleotide sequence comprising nucleotides #1, 232, 406, 427, 751 or 796 to #1059 or 1098 of SEQ ID NO:3. Accordingly, active human BMP-18 proteins include those comprising amino acids #31 135, xe2x88x9258, 1, 8, 116 or 131 to #218 or 231 of SEQ ID NO: 4.
The BMP-17 or BMP-18 proteins of the present invention, include polypeptides having a molecular weight of about 24.8 to 25.6 kD in monomeric form, said polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, respectively, and having the ability to induce the formation of cartilage and/or bone and/or other connective tissue in the Rosen-Modified Sampath-Reddi ectopic implant assay, described in the examples.
The BMP-17 or BMP-18 proteins recovered from the culture medium are purified by isolating them from other proteinaceous materials from which they are co-produced and from other contaminants present. BMP-17 or BMP-18 proteins may be characterized by the ability to induce the formation of cartilage and/or bone and/or other connective tissue and other tissue repair and differentiation, for example, in the rat bone formation assay described below. In addition, BMP-17 or BMP-18 proteins may be further characterized by their effects upon the growth and/or differentiation of embryonic cells and/or stem cells. Thus, the proteins or compositions of the present invention may be characterized by the embryonic stem cell assay described below.
The BMP-17 or BMP-18 proteins provided herein also include factors encoded by the sequences similar to those of SEQ ID NO: 1 or SEQ ID NO: 3, but into which modifications or deletions are naturally provided (e.g. allelic variations in the nucleotide sequence which may result in amino acid changes in the polypeptide) or deliberately engineered. For example, synthetic polypeptides may wholly or partially duplicate continuous sequences of the amino acid residues of SEQ ID NO:2 or SEQ ID NO: 4. These sequences, by virtue of sharing primary, secondary, or tertiary structural and conformational characteristics with bone growth factor polypeptides of SEQ ID NO: 2 or SEQ ID NO: 4 may possess biological properties in common therewith. It is know, for example that numerous conservative amino acid substitutions are possible without significantly modifying the structure and conformation of a protein, thus maintaining the biological properties as well. For example, it is recognized that conservative amino acid substitutions may be made among amino acids with basic side chains, such as lysine (Lys or K), arginine (Arg or R) and histidine (His or H); amino acids with acidic side chains, such as aspartic acid (Asp or D) and glutamic acid (Glu or E); amino acids with uncharged polar side chains, such as asparagine (Asn or N), glutamine (Gln or Q), serine (Ser or S), threonine (Thr or T), and tyrosine (Tyr or Y); and amino acids with nonpolar side chains, such as alanine (Ala or A), glycine (Gly or G), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), proline (Pro or P), phenylalanine (Phe or F), methionine (Met or M), tryptophan (Trp or W) and cysteine (Cys or C). Thus, these modifications and deletions of the native BMP-17 or BMP-18 may be employed as biologically active substitutes for naturally-occurring BMP-17 or BMP-18 and other polypeptides in therapeutic processes. It can be readily determined whether a given variant of BMP-17 or BMP-18 maintains the biological activity of BMP-17 or BMP-18 by subjecting both BMP-17 or BMP-18 and the variant of BMP-17 or BMP-18 to the assays described in the examples.
Other specific mutations of the sequences of BMP-17 or BMP-18 proteins described herein involve modifications of glycosylation sites. These modifications may involve O-linked or N-linked glycosylation sites. For instance, the absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at asparagine-linked glycosylation recognition sites. The asparagine-linked glycosylation recognition sites comprise tripeptide sequences which are specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or asparagine-X-serine, where X is usually any amino acid. A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence. Additionally, bacterial expression of BMP-17 and BMP-18-related protein will also result in production of a non-glycosylated protein, even if the glycosylation sites are left unmodified.
The present invention also encompasses the novel DNA sequences, free of association with DNA sequences encoding other proteinaceous materials, and coding for expression of BMP-17 and BMP-18 proteins. These DNA sequences include those depicted in SEQ ID NO:1 or SEQ ID NO: 3 in a 5xe2x80x2 to 3xe2x80x2 direction and those sequences which hybridize thereto under stringent hybridization washing conditions [for example, 0.1xc3x97SSC, 0.1% SDS at 65xc2x0 C.; see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to 389] and encode a protein maintaining one or more of the activities disclosed herein for BMP-17 or BMP-18. These DNA sequences also include those which comprise the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and those which hybridize thereto under stringent hybridization conditions and encode a protein which maintain the other activities disclosed for BMP-17 or BMP-18.
Similarly, DNA sequences which code for BMP-17 or BMP-18 polypeptides coded for by the sequences of SEQ ID NO: 1 or SEQ ID NO:3, respectively, or BMP-17 or BMP-18 polypeptides which comprise the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, but which differ in codon sequence due to the degeneracies of the genetic code or allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) also encode the novel factors described herein. Variations in the DNA sequences of SEQ ID NO: 1 or SEQ ID NO: 3 which are caused by point mutations or by induced modifications (including insertion, deletion, and substitution) to enhance the activity, half-life or production of the polypeptides encoded are also encompassed in the invention.
Another aspect of the present invention provides a novel method for producing BMP-17 or BMP-18 polypeptides. The method of the present invention involves culturing a suitable cell line, which has been transformed with a DNA sequence encoding a BMP-17 or BMP-18 polypeptide of the invention, under the control of known regulatory sequences. The transformed host cells are cultured and the BMP-17 or BMP-18 polypeptides recovered and purified from the culture medium. The purified polypeptides are substantially free from other proteins with which they are co-produced as well as from other contaminants.
Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO). The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1650 (1985) or Howley et al, U.S. Pat. No. 4,419,446. Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey COS-1 cell line. The mammalian cell CV-1 may also be suitable.
Bacterial cells may also be suitable hosts. For example, the various strains of E. coli (e.g., HB101, MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method. For expression of the protein in bacterial cells, DNA encoding the propeptide of BMP-17 or BMP-18 is generally not necessary.
Many strains of yeast cells known to those skilled in the art may also be available as host cells for expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein.
Another aspect of the present invention provides vectors for use in the method of expression of these novel BMP-17 or BMP-18 polypeptides. Preferably the vectors contain the full novel DNA sequences described above which encode the novel factors of the invention. Additionally, the vectors contain appropriate expression control sequences permitting expression of the BMP-17 or BMP-18 polypeptide sequences. Alternatively, vectors incorporating modified sequences as described above are also embodiments of the present invention. Additionally, the sequence of SEQ ID NO:1 or SEQ ID NO: 3 or other sequences encoding BMP-17 or BMP-18 polypeptides could be manipulated to express a mature BMP-17 or BMP-18 polypeptide by deleting BMP-17 or BMP-18 propeptide sequences and replacing them with sequences encoding the complete propeptides of other BMP proteins or members of the TGF-xcex2 superfamily. Thus, the present invention includes chimeric DNA molecules encoding a propeptide from a member of the TGF-xcex2 superfamily linked in correct reading frame to a DNA sequence encoding a BMP-17 or BMP-18 polypeptide.
The vectors may be employed in the method of transforming cell lines and contain selected regulatory sequences in operative association with the DNA coding sequences of the invention which are capable of directing the replication and expression thereof in selected host cells. Regulatory sequences for such vectors are known to those skilled in the art and may be selected depending upon the host cells. Such selection is routine and does not form part of the present invention.
A protein of the present invention, which induces cartilage and/or bone and/or other connective tissue formation in circumstances where such tissue is not normally formed, has application in the healing of bone fractures and cartilage or other connective tissue defects in humans and other animals. Such a preparation employing a BMP-17 or BMP-18 polypeptide may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery. A BMP-17 or BMP-18-related polypeptide may be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells, and may also support the regeneration of the periodontal ligament and attachment apparatus, which connects bone and teeth. BMP-17 or BMP-18 polypeptides of the invention may also be useful in the treatment of osteoporosis. A variety of osteogenic, cartilage-inducing and bone inducing factors have been described. See, e.g., European patent applications 148,155 and 169,016 for discussions thereof.
The proteins of the invention may also be used in wound healing and related tissue repair. The types of wounds include, but are not limited to burns, incisions and ulcers. (See, e.g. PCT Publication WO84/01106 for discussion of wound healing and related tissue repair). It is further contemplated that proteins of the invention may increase neuronal, astrocytic and glial cell survival and therefore be useful in transplantation and treatment of conditions exhibiting a decrease in neuronal survival and repair. The proteins of the invention may further be useful for the treatment of conditions related to other types of tissue, such as nerve, epidermis, muscle, and other organs such as liver, lung, epithelium, brain, spleen, cardiac, pancreas and kidney tissue. The proteins of the present invention may further be useful for the treatment of relatively undifferentiated cell populations, such as embryonic cells, or stem cells, to enhance growth and/or differentiation of the cells. The proteins of the present invention may also have value as a dietary supplement, or as a component of cell culture media. For this use, the proteins may be used in intact form, or may be predigested to provide a more readily absorbed supplement.
The proteins of the invention may also have other useful properties characteristic of the TGF-xcex2 superfamily of proteins. Such properties include angiogenic, chemotactic and/or chemoattractant properties, and effects on cells including induction of collagen synthesis, fibrosis, differentiation responses, cell proliferative responses and responses involving cell adhesion, migration and extracellular matrices. These properties make the proteins of the invention potential agents for wound healing, reduction of fibrosis and reduction of scar tissue formation.
When dimerized as a homodimer or as a heterodimer with other BMPs, with other members of the TGF-xcex2 superfamily of proteins, or with inhibin-xcex1 proteins or inhibin-xcex2 proteins, the BMP-17 or BMP-18 heterodimer is expected to demonstrate effects on the production of follicle stimulating hormone (FSH), as described further herein. It is recognized that FSH stimulates the development of ova in mammalian ovaries (Ross et al., in Textbook of Endocrinology, ed. Williams, p. 355 (1981) and that excessive stimulation of the ovaries with FSH will lead to multiple ovulations. FSH is also important in testicular function. Thus, BMP-17 or BMP-18 may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in mammals. BMP-17 or BMP-18 may also be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No. 4,798,885. BMP-17 or BMP-18 may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs. It is further contemplated that BMP-17 or BMP-18 may be useful in modulating hematopoiesis by inducing the differentiation of erythroid cells [see, e.g., Broxmeyer et al, Proc. Natl. Acad. Sci. USA, 85:9052-9056 (1988) or Eto et al, Biochem. Biophys. Res. Comm., 142:1095-1103 (1987)], for suppressing the development of gonadal tumors [see, e.g., Matzuk et al., Nature, 360:313-319 (1992)] or for augmenting the activity of bone morphogenetic proteins [see, e.g., Ogawa et al., J. Biol. Chem., 267:14233-14237 (1992)].
BMP-17 and BMP-18 proteins may be further characterized by their ability to modulate the release of follicle stimulating hormone (FSH) in established in vitro bioassays using rat anterior pituitary cells as described [see, e.g., Vale et al, Endocrinology, 91:562-572 (1972); Ling et al., Nature, 321:779-782 (1986) or Vale et al., Nature, 321:776-779 (1986)]. It is contemplated that the BMP-17 or BMP-18 protein of the invention, when composed as a heterodimer with inhibin xcex1 or inhibin xcex2 chains, will exhibit regulatory effects, either stimulatory or inhibitory, on the release of follicle stimulating hormone (FSH), from anterior pituitary cells as described [Ling et al., Nature, 321:779-782 (1986) or Vale et al., Nature, 321:776-779 (1986); Vale et al, Endocrinology, 91:562-572 (1972). Therefore, depending on the particular composition, it is expected that the BMP-17 or BMP-18 protein of the invention may have contrasting and opposite effects on the release of follicle stimulating hormone (FSH) from the anterior pituitary.
Activin A (the homodimeric composition of inhibin xcex2A) has been shown to have erythropoietic-stimulating activity [see e.g. Eto et al., Biochem. Biophys. Res. Commun., 142:1095-1103 (1987) and Murata et al., Proc. Natl. Acad. Sci. U.S.A., 85:2434-2438 (1988) and Yu et al., Nature, 330:765-767 (1987)]. It is contemplated that the BMP-17 and -18 proteins of the invention may have a similar erythropoietic-stimulating activity. This activity of the BMP-17 and BMP-18 proteins may be further characterized by the ability of the BMP-17 and BMP-18 proteins to demonstrate erythropoietin activity in the biological assay performed using the human K-562 cell line as described by [Lozzio et al., Blood, 45:321-334 (1975) and U.S. Pat. No. 5,071,834].
A further aspect of the invention is a therapeutic method and composition for repairing fractures and other conditions related to cartilage and/or bone and/or other connective tissue defects or periodontal diseases. The invention further comprises therapeutic methods and compositions for wound healing and tissue repair. Such compositions comprise a therapeutically effective amount of at least one of the BMP-17 or BMP-18-related proteins of the invention in admixture with a pharmaceutically acceptable vehicle, carrier or matrix. It is further contemplated that compositions of the invention may increase neuronal survival and therefore be useful in transplantation and treatment of conditions exhibiting a decrease in neuronal survival. Compositions of the invention may further include at least one other therapeutically useful agent, such as members of the TGF-xcex2 superfamily of proteins, which includes the BMP proteins BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed for instance in U.S. Pat. No. 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publication WO91/18098; BMP-9, disclosed in U.S. Pat. No. 5,661,007; BMP-10, disclosed in U.S. Pat. No. 5,637,480; BMP-11, disclosed in U.S. Pat. No. 5,639,638; BMP-12 or BMP-13, disclosed in U.S. Pat. No. 5,658,882; or BMP-15, disclosed in U.S. Pat. No. 5,635,372; or BMP-16, disclosed in co-pending patent application Ser. No. 08/715,202. Other compositions which may also be useful include Vgr-2, and any of the growth and differentiation factors [GDFs], including those described in PCT applications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others. Also useful in the present invention may be BIP, disclosed in WO94/01557; HP00269, disclosed in JP Publication number: 7-250688; and MP52, disclosed in PCT application WO93/16099. The disclosures of the above applications are hereby incorporated by reference herein.
It is expected that human BMP-17 and BMP-18 protein may exist in nature as monomers, or as homodimers or heterodimers. To promote the formation of dimers of BMP-17 or BMP-18 and useful proteins with increased stability, one can genetically engineer the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 to provide one or more additional cysteine residues to increase potential dimer formation. The resulting DNA sequence would be capable of producing a xe2x80x9ccysteine added variantxe2x80x9d of BMP-17 or BMP-18. In a preferred embodiment, one would engineer the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 so that one or more codons may be altered to a nucleotide triplet encoding a cysteine residue, such as TGT or TGC. Alternatively, one can produce xe2x80x9ccysteine added variantsxe2x80x9d of BMP-17 or BMP-18 protein by altering the sequence of the protein at the amino acid level by altering one or more amino acid residues of SEQ ID NO:2 or SEQ ID NO: 4 to cysteine. Production of xe2x80x9ccysteine added variantsxe2x80x9d of proteins is described in U.S. Pat. No. 5,166,322, the disclosure of which is hereby incorporated by reference. In preferred embodiments, the glutamic acid residue at position #173 of SEQ. ID NO: 2 or position #180 of SEQ. ID NO: 4 is replaced by a cysteine residue.
It is expected that the proteins of the invention may act in concert with or perhaps synergistically with other related proteins and growth factors. Further therapeutic methods and compositions of the invention therefore comprise a therapeutic amount of at least one BMP-17 or BMP-18 protein of the invention with a therapeutic amount of at least one other member of the TGF-xcex2 superfamily of proteins, such as the BMP proteins disclosed in the applications described above. Such combinations may comprise separate molecules of the BMP proteins or heteromolecules comprised of different BMP moieties. For example, a method and composition of the invention may comprise a disulfide linked dimer comprising a BMP-17 or BMP-18 protein subunit and a subunit from one of the xe2x80x9cBMPxe2x80x9d proteins described above. Thus, the present invention includes a purified BMP-17 or BMP-18-related polypeptide which is a heterodimer wherein one subunit comprises the amino acid sequence from amino acid #1 to amino acid #224 of SEQ ID NO:2 or #1 to #231 of SEQ ID NO:4, and one subunit comprises an amino acid sequence for a bone morphogenetic protein selected from the group consisting of BMP-2, BMP-3, BMP4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, or BMP-16. A further embodiment may comprise a heterodimer of BMP-17 or BMP-18-related moieties, for example a heterodimer of human BMP-17 and human BMP-18 or of human BMP-17 and the murine Letfy protein, which is a homologue of human BMP-17 and BMP-18. Further, BMP-17 or BMP-18 protein maybe combined with other agents beneficial to the treatment of the bone and/or cartilage and/or other connective tissue defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-xcex1 and TGF-xcex2 ), activins, inhibins, and k-fibroblast growth factor (kFGF), parathyroid hormone (PTH), parathyroid hormone related peptide (PTHrP), leukemia inhibitory factor (LIB/HILA/DA), insulin-like growth factors (IGF-I and IGF-II). Portions of these agents may also be used in compositions of the present invention. The preparation and formulation of such physiologically acceptable protein compositions, having due regard to pH, isotonicity, stability and the like, is within the skill of the art. The therapeutic compositions are also presently valuable for veterinary applications due to the lack of species specificity in BMP proteins. Particularly domestic animals and thoroughbred horses in addition to humans are desired patients for such treatment with the BMP-17 or BMP-18 proteins of the present invention.
The therapeutic method includes administering the composition topically, systemically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or other connective tissue or other tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than the BMP-17 or BMP-18 proteins which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the BMP composition in the methods of the invention.
Preferably for bone and/or cartilage and/or other connective tissue formation, the composition includes a matrix capable of delivering BMP-17 or BMP-18-related or other BMP proteins to the site of bone and/or cartilage and/or other connective tissue damage, providing a structure for the developing bone and cartilage and other connective tissue and optimally capable of being resorbed into the body. The matrix may provide slow release of BMP-17 or BMP-18 protein and/or other bone inductive protein, as well as proper presentation and appropriate environment for cellular infiltration. Such matrices may be formed of materials presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the BMP-17 or BMP-18 compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
The dosage regimen will be determined by the attending physician considering various factors which modify the action of the BMP-17 or BMP-18 protein, e.g. amount of bone or other tissue weight desired to be formed, the site of bone or tissue damage, the condition of the damaged bone tissue, the size of a wound, type of damaged tissue, the patient""s age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and the types of BMP proteins in the composition. Generally, systemic or injectable administration will be initiated at a dose which is minimally effective, and the dose will be increased over a preselected time course until a positive effect is observed. Subsequently, incremental increases in dosage will be made limiting such incremental increases to such levels that produce a corresponding increase in effect, while taking into account any adverse affects that may appear. The addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.
Progress can be monitored by periodic assessment of bone or tissue growth and/or repair. The progress can be monitored, for example, x-rays, histomorphometric determinations and tetracycline labeling.
The following examples illustrate practice of the present invention with respect to human BMP-17 and human BMP-18 and other BMP-17 and BMP-18-related proteins. The skilled artisan will recognize that numerous variations and modifications are possible. These variations and modifications constitute part of the present invention.